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Current Research and Scholarly Interests

Our laboratory examines apoptotic and cell signaling pathways in the lung with a focus on fibrotic lung disease and lung cancer. We are studying TGF beta signaling in lung cells which regulates fibroblast and epithelial activation. We are also studying non-canonical functions of TERC and Tert in regulation of transcription in cancer cells and activated lung epithelium. The analysis of TERC lncRNA regulation of transcription ,which is done in collaboration with Dr. Howard Chang. We also are involved in trials of novel therapeutics for patients with pulmonary fibrosis.

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Abstract

Interstitial lung disease (ILD) is commonly encountered in patients with connective tissue diseases (CTD). Besides the lung parenchyma, the airways, pulmonary vasculature and structures of the chest wall may all be involved, depending on the type of CTD. As a result of this so-called multi-compartment involvement, airflow limitation, pulmonary hypertension, vasculitis and extrapulmonary restriction can occur alongside fibro-inflammatory parenchymal abnormalities in CTD. Rheumatoid arthritis (RA), systemic sclerosis (SSc), poly-/dermatomyositis (PM/DM), Sjögren's syndrome (SjS), systemic lupus erythematosus (SLE), and undifferentiated (UCTD) as well as mixed connective tissue disease (MCTD) can all be associated with the development of ILD. Non-specific interstitial pneumonia (NSIP) is the most commonly observed histopathological pattern in CTD-ILD, but other patterns including usual interstitial pneumonia (UIP), organizing pneumonia (OP), diffuse alveolar damage (DAD) and lymphocytic interstitial pneumonia (LIP) may occur. Although the majority of patients with CTD-ILD experience stable or slowly advancing ILD, a small yet significant group exhibits a more severe and progressive course. Randomized placebo-controlled trials evaluating the efficacy of immunomodulatory treatments have been conducted only in SSc-associated ILD. However, clinical experience suggests that a handful of immunosuppressive medications are potentially effective in a sizeable portion of patients with ILD caused by other CTDs. In this manuscript, we review the clinical characteristics and management of the most common CTD-ILDs.

Abstract

To compare survival of patients with connective tissue disease-associated interstitial lung disease (CTD-ILD) versus idiopathic pulmonary fibrosis (IPF) and patients with systemic sclerosis-associated ILD (SSc-ILD) versus other CTD-ILD followed at our center.We used the Stanford ILD database, which contains prospectively collected information on patients with ILD evaluated at our tertiary care center from 2002 to 2009. Survival at last followup from time of ILD diagnosis was calculated using the Kaplan-Meier estimator. Prognostic factors for survival in the overall cohort (IPF and CTD-ILD) and in the CTD-ILD group were identified with univariate and multivariate Cox regression models.Of 427 patients with ILD, 148 (35%) had IPF and 76 (18%) had CTD-ILD at the baseline visit. The cumulative incidence of CTD was 4%. After a median followup of 4 years, 67 patients (36.4%) had died and 4 (2.2%) were lost to followup. Patients with IPF (n = 122) and CTD-ILD (n = 62) experienced similar survival rates (5-year survival about 50%). Patients with SSc-ILD (n = 24) experienced better survival than those with other CTD-ILD (n = 38), with 1-year, 3-year, and 5-year survival rates of 100%, 90%, and 77%, respectively, versus 78%, 42%, and 38% (p = 0.01). The presence of SSc in patients with CTD-ILD decreased the risk of death by > 80% even after correcting for age at ILD diagnosis, sex, and ethnicity (HR = 0.17, 95% CI 0.04-0.83).Survival in patients with SSc-ILD was better than in patients with other CTD-ILD, potentially related to routine screening for and early detection of ILD in patients with SSc at our center.

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive and medically refractory lung disease with a grim prognosis. Although the etiology of IPF remains perplexing, abnormal adaptive immune responses are evident in many afflicted patients. We hypothesized that perturbations of human leukocyte antigen (HLA) allele frequencies, which are often seen among patients with immunologic diseases, may also be present in IPF patients.HLA alleles were determined in subpopulations of IPF and normal subjects using molecular typing methods. HLA-DRB1*15 was over-represented in a discovery cohort of 79 Caucasian IPF subjects who had lung transplantations at the University of Pittsburgh (36.7%) compared to normal reference populations. These findings were prospectively replicated in a validation cohort of 196 additional IPF subjects from four other U.S. medical centers that included both ambulatory patients and lung transplantation recipients. High-resolution typing was used to further define specific HLA-DRB1*15 alleles. DRB1*1501 prevalence in IPF subjects was similar among the 143 ambulatory patients and 132 transplant recipients (31.5% and 34.8%, respectively, p?=?0.55). The aggregate prevalence of DRB1*1501 in IPF patients was significantly greater than among 285 healthy controls (33.1% vs. 20.0%, respectively, OR 2.0; 95%CI 1.3-2.9, p?=?0.0004). IPF patients with DRB1*1501 (n?=?91) tended to have decreased diffusing capacities for carbon monoxide (DL(CO)) compared to the 184 disease subjects who lacked this allele (37.8±1.7% vs. 42.8±1.4%, p?=?0.036).DRB1*1501 is more prevalent among IPF patients than normal subjects, and may be associated with greater impairment of gas exchange. These data are novel evidence that immunogenetic processes can play a role in the susceptibility to and/or manifestations of IPF. Findings here of a disease association at the HLA-DR locus have broad pathogenic implications, illustrate a specific chromosomal area for incremental, targeted genomic study, and may identify a distinct clinical phenotype among patients with this enigmatic, morbid lung disease.

Abstract

Idiopathic pulmonary fibrosis (IPF) is a poorly understood progressive disease characterized by the accumulation of scar tissue in the lung interstitium. A hallmark of the disease is areas of injury to type II alveolar epithelial cells with attendant accumulation of fibroblasts in areas called fibroblastic foci. In an effort to better characterize the lung fibroblast phenotype in IPF patients, we isolated fibroblasts from patients with IPF and looked for activation of signaling proteins, which could help explain the exaggerated fibrogenic response in IPF. We found that IPF fibroblasts constitutively expressed increased basal levels of SPARC, plasminogen activator inhibitor-1 (PAI-1), and active beta-catenin compared with control cells. Control of basal PAI-1 expression in IPF fibroblasts was regulated by SPARC-mediated activation of Akt, leading to inhibition of glycogen synthase kinase-3beta and activation of beta-catenin. Additionally, IPF fibroblasts (but not control fibroblasts) were resistant to plasminogen-induced apoptosis and were sensitized to plasminogen-mediated apoptosis by inhibition of SPARC or beta-catenin. These findings uncover a newly discovered regulatory pathway in IPF fibroblasts that is characterized by elevated SPARC, giving rise to activated beta-catenin, which regulates expression of downstream genes, such as PAI-1, and confers an apoptosis-resistant phenotype. Disruption of this pathway may represent a novel therapeutic target in IPF.

Abstract

We studied the role of Bim, a pro-apoptotic BCL-2 family member in Airborne particulate matter (PM 2.5 microm)-induced apoptosis in alveolar epithelial cells (AEC). PM induced AEC apoptosis by causing significant reduction of mitochondrial membrane potential and increase in caspase-9, caspase-3 and PARP-1 activation. PM upregulated pro-apoptotic protein Bim and enhanced translocation of Bim to the mitochondria. ShRNABim blocked PM-induced apoptosis by preventing activation of the mitochondrial death pathway suggesting a role of Bim in the regulation of mitochondrial pathway in AEC. Accordingly, we provide the evidence that Bim mediates PM-induced apoptosis via mitochondrial pathway.

Abstract

Pulmonary intravascular bronchoalveolar tumor (IVBAT) also recognized as pulmonary epithelioid hemangioendothelioma, is a rare malignant vascular tumor of unknown etiology. IVBAT is a tumor of multicentric origin and the lungs are rarely involved, with only about 60 cases of pulmonary IVBAT described in the literature. The prognosis is unpredictable, with life expectancy ranging from 1 to 15 years. We report an unusual case of pulmonary IVBAT that recurred in the lung with metastasis to the mediastinum.

Abstract

Idiopathic pulmonary fibrosis is a progressive, fatal disease with no known efficacious therapy.In a double-blind, multinational trial, we randomly assigned 330 patients with idiopathic pulmonary fibrosis that was unresponsive to corticosteroid therapy to receive subcutaneous interferon gamma-1b or placebo.Over a median of 58 weeks, interferon gamma-1b therapy did not significantly affect the primary end point of progression-free survival, defined as the time to disease progression or death, and no significant treatment effect was observed on measures of lung function, gas exchange, or the quality of life. Ten percent of patients in the interferon gamma-1b group died, as compared with 17 percent of patients in the placebo group (P=0.08). Treatment with interferon gamma-1b was associated with more frequent constitutional symptoms. However, the rates of treatment adherence and premature discontinuation of treatment were similar in the two groups. More pneumonias were reported among patients in the interferon gamma-1b group, but the incidence of severe or life-threatening respiratory tract infections was similar in the two groups.In a well-defined population of patients with idiopathic pulmonary fibrosis, interferon gamma-1b did not affect progression-free survival, pulmonary function, or the quality of life. Owing to the size and duration of the trial, a clinically significant survival benefit could not be ruled out.

Abstract

Treatment of solid tumors with combinations of chemotherapeutic agents has not led to significant increases in long-term survival. Recent studies support a role for inhibitors of checkpoint arrest as a means to enhance the cytotoxicity of chemotherapy. We have shown previously that triptolide (PG490), an oxygenated diterpene derived from a Chinese medicinal plant, induces apoptosis in cultured tumor cells and sensitizes tumor cells to topoisomerase inhibitors by blocking p53-mediated induction of p21. Here we extend our studies to a tumor xenograft model and evaluate the efficacy and safety of PG490-88 (14-succinyl triptolide sodium salt), a water-soluble prodrug of PG490. We also look at the combination of PG490 or PG490-88 with CPT-11, a topoisomerase I inhibitor, in cultured cells and in the tumor xenograft model. We show that PG490-88 is a safe and potent antitumor agent when used alone, causing tumor regression of lung and colon tumor xenografts. We also show that PG490-88 acts in synergy with CPT-11 to cause tumor regression. A phase I trial of PG490-88 for solid tumors began recently and safety and optimal dosing data should accrue within the next 12 months. Our findings that PG490-88 causes tumor regression and that it acts in synergy with DNA-damaging chemotherapeutic agents suggest a role as an antineoplastic agent and chemosensitizer for the treatment of patients with solid tumors.

Abstract

TNF-related apoptosis-inducing ligand (TRAIL/Apo- 2L), a newly identified member of the TNF family promotes apoptosis by binding to the transmembrane receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). TRAIL known to activate NF-kappaB in number of tumor cells including A549 (wt p53) and NCI-H1299 (null p53) lung cancer cells exerts relatively selective cytotoxic affects to the human tumor cell lines without much effect on the normal cells. We set out to identify an agent that would sensitize lung cancer cells to TRAIL-induced apoptosis through inhibition of NF-kappaB activation. We found that triptolide, an oxygenated diterpene extracted and purified from the Chinese herb Tripterygium wilfordii sensitized A549 and NCI-H1299 cells to TRAIL-induced apoptosis through inhibition of NF-kappaB activation. Pretreatment with MG132 which is a well-known NF-kappaB inhibitor by blocking degradation of IkappaBalpha also greatly sensitized lung cancer cells to TRAIL-induced apoptosis. Triptolide did not block DNA binding of NF-kappaB activated by TRAIL as in the case of TNF-alpha. It has been already proven that triptolide blocks transactivation of p65 which plays a key role in NF-kappaB activation. These observations suggest that triptolide may be a potentially useful drug to enhance TRAIL-induced tumor killing in lung cancer.

Abstract

The current treatment of obliterative bronchiolitis in lung transplant recipients is sub-optimal. Triptolide is a novel immunosuppressant that has a mechanism of action distinct from currently available immunosuppressants, including induction of T-cell apoptosis, blockade of fibroblast proliferation/maturation and inhibition of transforming growth factor-beta (TGF-beta) mRNA production. We hypothesized that triptolide may be helpful in blocking obliterative airway disease in lung transplant recipients. We investigated the effect of PG490-88, a water-soluble derivative of triptolide, in a mouse heterotopic tracheal allograft model of obliterative airway disease. We show that PG490-88 attenuates airway obliteration in this model and inhibits accumulation of inflammatory cells, and therefore may have preventive or therapeutic benefits for patients with obliterative airway disease (OAD) following lung transplantation.

Abstract

Lung cancer is the result of molecular changes that occur in the cell, resulting in the deregulation of pathways which control normal cellular growth, differentiation, and apoptosis. Several of these pathways contain well-characterized proto-oncogenes and tumor suppressor genes which are found to be mutated or have abnormal expression patterns in lung cancer. The molecular changes that characterize lung cancer are complex, but it is known that cigarette smoking causes most squamous cell and small-cell carcinomas. However, the association between cigarette smoke and adenocarcinoma is less clear. Environmental factors, such as air pollutants, radon, and asbestos, likely contribute to the development of lung cancer. In this review, we discuss the major molecular abnormalities in lung cancer with a review of recent studies that begin to decipher the role that different tumor suppressor genes and oncogenes play in the pathogenesis of lung cancer. Also, we highlight the research that has identified new genes which may play a role in lung cancer pathogenesis or progression.

Abstract

Obliterative bronchiolitis (OB) is a dreaded and frequent complication of lung transplantation with a poorly understood immunopathogenesis. To further evaluate disease mechanisms, we used T cell antigen receptor (TCR) beta-chain variable region RNase protection assays, after polymerase chain reaction amplification of TCR cDNA, to quantitate circulating CD4(+) and CD8(+) repertoires of transplant recipients with OB or no evidence of rejection (NER). All six recipients with OB had markedly abnormal CD4 expansions (2.5 +/- 0.5 expansions/recipient) attributable to oligoclonal proliferations. Only two of six recipients with NER had a single, much lesser, CD4(+) abnormality each (p < 0.01). Moreover, one of these patients developed OB shortly thereafter, and the other NER abnormality may have predated transplantation. In contrast, CD8(+) expansions were common in both recipient populations. Findings of CD4(+) expansions had 100% sensitivity and 80% specificity for the presence or imminent development of OB. These data suggest proliferations of CD4(+) T cells are important in OB pathogenesis, and these are most likely part of a major histocompatibility complex Class II-dependent process of indirect alloantigen presentation. These CD4(+) clones are likely to have facultative helper functions for the multiple and diverse immune processes that have been implicated in OB. Furthermore, the close association of CD4(+) expansions with OB raises possibilities of development of novel diagnostic and therapeutic approaches.

Abstract

The global gene expression profiles for 67 human lung tumors representing 56 patients were examined by using 24,000-element cDNA microarrays. Subdivision of the tumors based on gene expression patterns faithfully recapitulated morphological classification of the tumors into squamous, large cell, small cell, and adenocarcinoma. The gene expression patterns made possible the subclassification of adenocarcinoma into subgroups that correlated with the degree of tumor differentiation as well as patient survival. Gene expression analysis thus promises to extend and refine standard pathologic analysis.

Abstract

In this study we evaluate the antifibrotic properties of PG-490-88, a water-soluble derivative of triptolide. Triptolide is an oxygenated diterpene that is derived from a traditional Chinese herb that has potent immunosuppressive and antitumor activity. We used the intratracheal bleomycin mouse model and found that PG490-88 inhibits fibrosis in the bleomycin group when given the same day or 5 days after bleomycin. PG490-88 also markedly reduced the number of myofibroblasts in the bleomycin treatment group. An enzyme-linked immunosorbent assay of transforming growth factor (TGF)-beta in the bronchoalveolar lavage fluid showed a significant decrease in TGF-beta in the PG490-88-treated groups compared to the bleomycin-treated group. Additionally, triptolide blocked bleomycin-induced increase in TGF-beta mRNA in cultured normal human lung fibroblasts. The efficacy of PG490-88 when administered late after bleomycin installation suggests a potential role in the treatment of idiopathic pulmonary fibrosis.

Abstract

Triptolide (PG490), a diterpene triepoxide, is a potent immunosuppressive agent extracted from the Chinese herb Tripterygium wilfordii. We have previously shown that triptolide blocks NF-kappaB activation and sensitizes tumor necrosis factor (TNF-alpha)-resistant tumor cell lines to TNF-alpha-induced apoptosis. We show here that triptolide enhances chemotherapy-induced apoptosis. In triptolide-treated cells, the expression of p53 increased but the transcriptional function of p53 was inhibited, and we observed a down-regulation of p21(waf1/cip1), a p53-responsive gene. The increase in levels of the p53 protein was mediated by enhanced translation of the p53 protein. Additionally, triptolide induced accumulation of cells in S phase and blocked doxorubicin-mediated accumulation of cells in G(2)/M and doxorubicin-mediated induction of p21. Our data suggest that triptolide, by blocking p21-mediated growth arrest, enhances apoptosis in tumor cells.

Abstract

Antigen presentation by lung macrophages/dendritic cells (DC) is thought to be important in obliterative bronchiolitis/bronchiolitis obliterans syndrome (OB/BOS), which severely limits survival post-lung transplantation. However, a recent study found minimal numbers of DC in lung allografts. We looked at numbers and phenotype of macrophages/DC in lung allografts using endobronchial biopsy (EBB) and transbronchial biopsy (TBB) from 22 lung transplant patients. Biopsies were stained with monoclonal markers of DC (CD1a, RFD1, and major histocompatibility complex [MHC] Class II), and "suppressor macrophages" (RFD1 and RFD7). Dendritic cells were also stained for the costimulatory molecules CD80 and CD86. Significantly greater numbers of DC/high-power field (HPF) were seen in biopsies when we defined DC using dendritic morphology and Class II MHC expression instead of CD1a expression. Dendritic cell numbers were significantly higher in eight patients with OB/BOS compared with 14 stable patients. Fifty percent of DC expressed CD86 and 20% expressed CD80. There was no difference in CD80 or CD86 expression between OB/BOS patients and stable patients. There was no correlation between DC numbers and presence or absence of acute rejection (AR), and/or cytomegalovirus (CMV) pneumonitis on current or prior biopsies. There were significantly more MHC Class II DC in EBB compared with TBB. We found minimal staining for lung macrophages capable of suppressing T-cell inflammation. We conclude that studies of lung allografts may underestimate DC numbers if relying on CD1a as the sole marker of DC. DC are increased in patients with OB/BOS compared with stable patients. EBB may be more important than TBB in looking for inflammatory changes of OB. DC expressing costimulatory molecules are present in lung allografts, and costimulatory pathway blockade may be useful in human lung allografts. Also, the absence of "suppressor" macrophages may increase susceptibility of human lung allografts to the rejection process.

Abstract

Interferon gamma (IFN-gamma) induces apoptosis in many tumor cell lines and sensitizes tumor cells to apoptosis by tumor necrosis factor family members. IFN-gamma induces the expression of many early response genes such as interferon regulatory factor-1 (IRF-1) by activation of signal transducer and activator of transcription (STAT) factor proteins. We found that ME180 cells became resistant to IFN-gamma-induced cell death after 4-5 passages in culture. These resistant cells were characterized by a loss of STAT1 expression and a loss of inducible IRF-1 expression. We describe for the first time the emergence of a STAT1-deficient ME180 cell line.

Abstract

Progress in the treatment of solid tumors has been slow and sporadic. The efficacy of conventional chemotherapy in solid tumors is limited because tumors frequently have mutations in the p53 gene. Also, chemotherapy only kills rapidly dividing cells. Members of the tumor necrosis factor (TNF) family, however, induce apoptosis regardless of the p53 phenotype. Unfortunately, the cytotoxicity of TNF-alpha is limited by its activation of NF-kappaB and activation of NF-kappaB is proinflammatory. We have identified a compound called PG490, that is composed of purified triptolide, which induces apoptosis in tumor cells and sensitizes tumor cells to TNF-alpha-induced apoptosis. PG490 potently inhibited TNF-alpha-induced activation of NF-kappaB. PG490 also blocked TNF-alpha-mediated induction of c-IAP2 (hiap-1) and c-IAP1 (hiap-2), members of the inhibitor of apoptosis (IAP) family. Interestingly, PG490 did not block DNA binding of NF-kappaB, but it blocked transactivation of NF-kappaB. Our identification of a compound that blocks TNF-alpha-induced activation of NF-kappaB may enhance the cytotoxicity of TNF-alpha on tumors in vivo and limit its proinflammatory effects.

Abstract

p120-ras GTPase-activating protein (rasGAP) associates with Ras and negatively regulates Ras signaling by stimulating the intrinsic rate of Ras GTPase activity. rasGAP also associates with other cellular signaling proteins which suggest that rasGAP may play a role in coordinating other signal transduction pathways. Disruption of rasGAP in vivo results in extensive apoptosis. Fas-mediated apoptosis results in the activation of caspases that cleave cellular substrates which are important for maintaining cytoplasmic and nuclear integrity. We show here that rasGAP is proteolytically cleaved by caspases early in Fas-induced apoptosis of Jurkat cells. rasGAP was also cleaved by DNA-damaging chemotherapeutic agents and TNF-related apoptosis inducing ligand (TRAIL), also known as Apo2L. Based on the size of the products generated by cleavage of deletion mutants of rasGAP we predict that cleavage of rasGAP occurs in the hydrophobic region and between the SH2(2) and ras-p21 interacting domain which would leave an intact ras-p21 interacting domain. Interestingly, cleavage of rasGAP in vitro enhanced rasGAP hydrolysis activity. Our results demonstrate that diverse apoptotic stimuli cause caspase-mediated cleavage of rasGAP early in apoptosis.

Abstract

Lung epithelium plays a central role in modulation of the inflammatory response and in lung repair. Airway epithelial cells are targets in asthma, viral infection, acute lung injury, and fibrotic lung disease. Activated T lymphocytes release cytokines such as interferon-gamma (IFN-gamma) that can cooperate with apoptotic signaling pathways such as the Fas-APO-1 pathway to induce apoptosis of damaged epithelial cells. We report that IFN-gamma alone and in combination with activation of the Fas pathway induced apoptosis in A549 lung epithelial cells. Interestingly, the corticosteroid dexamethasone was the most potent inhibitor of IFN-gamma- and IFN-gamma plus anti-Fas-induced apoptosis. IFN-gamma induced expression of an effector of apoptosis, the cysteine protease interleukin-1 beta-converting enzyme, in A549 cells. Dexamethasone, in contrast, induced expression of an inhibitor of apoptosis, human inhibitor of apoptosis (hIAP-1), also known as cIAP2. We suggest that the inhibition of epithelial cell apoptosis by corticosteroids may be one mechanism by which they suppress the inflammatory response.

Abstract

Apoptotic cells undergo characteristic morphological changes that include detachment of cell attachment from the substratum and loss of cell-cell interactions. Attachment of cells to the extracellular matrix and to other cells is mediated by integrins. The interactions of integrins with the extracellular matrix activates focal adhesion kinase (FAK) and suppresses apoptosis in diverse cell types. Members of the tumor necrosis family such as Fas and Apo-2L, also known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), induce apoptosis in both suspension and adherent cells through the activation of caspases. These caspases, when activated, cleave substrates that are important for the maintenance of nuclear and membrane integrity. In this study, we show that FAK is sequentially cleaved into two different fragments early in Apo-2L-induced apoptosis. We also demonstrate that FAK cleavage is mediated by caspases and that FAK shows unique sensitivity to different caspases. Our results suggest that disruption of FAK may contribute to the morphological changes observed in apoptotic suspension and adherent cells.

Abstract

Obliterative bronchiolitis is the major cause of long-term morbidity and mortality in heart-lung and lung transplant recipients. There is presently no completely effective therapy for the treatment of obliterative bronchiolitis. We have examined the effects of rapamycin (RPM) on the development of obliterative airway disease in murine recipients of heterotopically transplanted allograft tracheas. In this model, an untreated allograft develops almost complete occlusion of the airway lumen with fibroblastic tissue and collagen scar by day 28 after transplantation. RPM administered intraperitoneally at the time of transplantation or even as late as day 14 after transplantation markedly inhibited obliteration of the airway lumen by fibroblastic tissue. Also, RPM significantly inhibited infiltration of the graft by macrophages. In the RPM-treated animals, the airway was reconstituted with an attenuated squamous epithelium rather than a normal pseudostratified epithelium. No adverse side effects were observed with RPM doses up to 12 mg/kg/ day. These findings suggest a potential role for RPM, perhaps in combination with cyclosporine, in preventing and treating obliterative bronchiolitis in heart-lung and lung allograft recipients.

Abstract

Airway epithelial cells modulate the inflammatory response in asthmatic, allergic and fibrotic lung diseases through the secretion of cytokines that regulate the movement and activation of inflammatory cells. Mast cells play an important role in the pathogenesis of these lung diseases. In this study we report that normal airway epithelial cells express stem cell factor which is a critical mediator of mast cell growth and differentiation and that transforming growth factor-beta inhibits secretion of stem cell factor by airway epithelial cells.

Abstract

It has been suggested that expression of the genes encoding the alpha 4/beta 1 integrin increases during wound healing of the cornea. As a first step in understanding the mechanisms required to stimulate alpha 4 gene expression during this process, we defined the minimal upstream sequence required to direct basal promoter activity for this gene. Using deletion analyses of the alpha 4 gene upstream sequence, we identified two functionally important negative regulatory elements. Dimethylsulfate (DMS) methylation interference assays provided evidence for the binding of a single nuclear protein to tandemly repeated homologous cis-acting elements (designated alpha 4.1 and alpha 4.2) from the alpha 4 basal promoter that share the core sequence 5'-GTGGGT-3'. The formation of a protection only at alpha 4.1 in DNase I footprinting suggested that it is the primary target element for the binding of nuclear proteins. Three distinct nuclear proteins bound a double-stranded oligonucleotide bearing the DNA sequence of alpha 4.1 to produce specific DNA-protein complexes (R1 to R3) in gel-shift assays, from which that producing R3 was identified as the protein yielding DNase I protection at alpha 4.1. Detailed mutational analysis of alpha 4.1 and alpha 4.2 indicated that both elements positively regulate gene expression in primary cultures of corneal epithelial cells and Jurkat tissue culture cells, which is consistent with the deletion analysis. However, when transiently transfected into pituitary GH4C1, the alpha 4.2 mutants yielded increased chloramphenicol acetyl transferase activity therefore demonstrating that these elements have the ability to function either as positive or negative regulators of gene transcription in a manner that is dependent on the type of cell transfected.

Abstract

alpha 4 integrins mediate cell-cell and cell-extracellular matrix interactions that are critical for maturation and function of the immune system as well as differentiation of skeletal muscle. Here we examine molecular mechanisms controlling the pattern of alpha 4 expression. The activity of constructs containing 5' deletion mutants of the alpha 4 gene promoter was compared in transfection assays into cell lines that express alpha 4 and cell lines that do not. The sequence between position -42 and -76 base pairs (bp) was required for efficient transcription in cells that express alpha 4, but it showed no activity in HeLa cells, which do not express alpha 4. Three binding sites for the Ets family of transcription factors are found in this region: two adjacent sites at positions -50 and -54 bp and a more 5' site at position -67 bp. Using a series of constructs containing deletions and mutations in this region, we found that the 3'-most site alone was sufficient for binding GA-binding protein alpha (GABP alpha)/GABP beta and for a low level of transcriptional activation. When all three sites were present, a second complex "a" was detected, which contains an unknown member of the Ets family. Formation of complex a was cell-type specific and correlated with a high level of transcription. Deletion of the 5'-most Ets site had no effect on binding to GABP alpha/GABP beta, but it eliminated a. Concomitant with this loss of a, a new Ets-1-containing complex "c" appeared. Complex c substituted efficiently for complex a in transcriptional activation. We conclude that although neither of the two 5'-most Ets sites alone binds nuclear protein, they appear to act as modulators which control the pattern of Ets proteins that bind the alpha 4 gene promoter. This arrangement of Ets sites, coupled with the tissue- and developmental-specific expression of Ets members, likely play a key role in defining the pattern of alpha 4 integrin.

Abstract

Interaction of alpha 4 integrins with vascular cell adhesion molecule-1 (VCAM-1) is classically important for immune function. However, we found recently that these receptors have a second role, in embryogenesis, where they mediate cell-cell interactions that are important for skeletal muscle differentiation. Here, we present evidence of an expanding role for these receptors in murine development. alpha 4 and VCAM-1 were found at embryonic sites of hematopoiesis, suggesting a role for these receptors during embryogenesis that parallels their hematopoietic function in adult bone marrow. During angiogenesis in the lung, alpha 4 and VCAM-1 were found on mesenchyme that gives rise to vascular endothelium and smooth muscle. alpha 4 persisted on the smooth muscle and the endothelium of newly forming vessels where it colocalized with its extracellular matrix ligand, fibronectin (FN). These patterns suggest several roles for alpha 4 integrins and their ligands in angiogenesis. alpha 4 was also found on neural crest derivatives where it colocalized with FN. alpha 4 was expressed selectively on cells in the dorsal root ganglia: it was apparent along ventral projections, but absent from dorsal projections, suggesting that alpha 4 integrins could be involved in defining neuronal fates. Although VCAM-1 was not expressed on most neural crest derivatives, it was found in the neural crest-derived outflow tract of the embryonic heart, where it colocalized with alpha 4. These results imply that alpha 4 integrins and their ligands could be important for migration or differentiation of neural crest. alpha 4 was also expressed on embryonic retina and FN was found on inductive mesenchyme surrounding the eye, suggesting a role for these proteins in eye development. Finally, based on their patterns of expression, we conclude that VCAM-1 only participates in a subset of interactions involving alpha 4 integrins, whereas FN appears to be the more general ligand.

Abstract

The integrin alpha 4 beta 1 and its counter receptor vascular cell adhesion molecule-1 (VCAM-1) mediate well-described cell-cell interactions that are critical for immune function. However, these receptors also mediate cell-cell interactions that are important for skeletal muscle differentiation. We have found that contrasting transcriptional mechanisms control their patterns of expression in the immune system and in muscle. Recent studies indicate that alpha 4 beta 1 and VCAM-1 are also expressed in a number of developing tissues, implying that these receptors have a general role in facilitating cell-cell interactions during development.

Abstract

Vascular cell adhesion molecule-1 (VCAM-1) was first identified as a protein that appears on the surface of endothelial cells after exposure to inflammatory cytokines. Through interaction with its integrin counter receptor VLA-4, VCAM-1 mediates cell-cell interactions important for immune function. We have cloned and begun characterization of the promoter for the VCAM-1 gene. In a series of transfection assays into human umbilical vein endothelial cells (HUVECs), we find that silencers between positions -1.641 kilobases and -288 base pairs restrict promoter activity, and that treatment with tumor necrosis factor-alpha overcomes this inhibition and activates the promoter through two NF kappa B sites located at positions -77 and -63 base pairs of the VCAM-1 gene. This responsiveness appears cell-specific since constructs containing the VCAM-1 NF kappa B sites are not responsive to tumor necrosis factor alpha in the T-cell line Jurkat. The two VCAM-1 NF kappa B sites, which differ slightly in their sequence, form distinct complexes in gel retardation assays, suggesting that they interact with different NF kappa B-site binding proteins. The distribution of these proteins could then control activity of the NF kappa B sites. We conclude that the pattern of VCAM-1 expression in HUVECs is controlled by a combination of these silencers and NF kappa B sites.

Abstract

Mammalian myogenesis is biphasic: primary myoblasts fuse to form primary myotubes, then secondary myoblasts align along the primary myotubes and form secondary myotubes, which comprise most of adult muscle. We provide evidence that an integrin (VLA-4) and its counter receptor (VCAM-1) have a role in secondary myogenesis. Both receptors are synthesized by cultured muscle cells: VLA-4 is induced as myotubes form, whereas VCAM-1 is present on myoblasts and myotubes. In vivo, both molecules are expressed at sites of secondary myogenesis, VLA-4 on primary and secondary myotubes, and VCAM-1 on secondary myoblasts and on regions of secondary myotubes apposed to primary myotubes. These patterns suggest that VLA-4-VCAM-1 interactions influence alignment of secondary myoblasts along primary myotubes and/or the fusion of secondary myoblasts. In support of the latter possibility, antibodies to VLA-4 or VCAM-1 inhibit myotube formation in culture.

Abstract

We examined expression of nuclear factor-1 (NF-1) in different cell lines. Expression was low or undetectable in T and B lymphocyte cell lines, whereas fibroblasts and other adherent cell lines generally had a relatively high level of NF-1 mRNA. In cell lines that did not express NF-1, gel retardation assays, nevertheless, indicated complexes between a protein or proteins and the NF-1 site. These complexes were less abundant than those formed with NF-1, they migrated more slowly, and they appeared as single species instead of the multiple species observed with NF-1. NF-1 site-binding proteins were compared in the fibrosarcoma cell line HT-1080 (expressed the highest level of NF-1 in our study) and the B cell line Raji (does not express NF-1). UV-crosslinking studies indicated that the NF-1 site-binding proteins in both cell lines were similar in size. Proteolytic clipping band shift assays suggested that the Raji protein and NF-1 share structural similarity in their DNA binding domains, but are distinct proteins. The NF-1 site mediated transcriptional stimulation in cell lines where NF-1 is expressed; however, this element did not affect transcription in cell lines that do not express NF-1, suggesting that the NF-1 site-binding protein in these cells is functionally distinct from NF-1.

Abstract

Fibronectin (FN) is an extracellular matrix protein that acts as a substrate for cell migration and adhesion during development. Cells adhere to FN through integral membrane proteins that are members of the integrin family of adhesion molecules. The interaction between cells and FN is important in a number of biologic processes, including gastrulation, hematopoietic differentiation, neural crest cell migration, cardiac development, branching morphogenesis in lung, wound healing, tumorigenesis, and metastasis. Expression of FN and its receptors is controlled by a number of hormones and growth factors as well as by tissue-specific factors. Here, the molecular aspects of how expression of these genes is controlled are reviewed, with particular emphasis on promoter regulator elements that modulate expression.

CHARACTERIZATION OF THE ALPHA-4 INTEGRIN GENE PROMOTERPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICARosen, G. D., BIRKENMEIER, T. M., Dean, D. C.1991; 88 (10): 4094-4098

Abstract

A cDNA for the alpha 4 chain of the alpha 4 beta 1 integrin was described previously [Takada, Y., Elices, M. J., Crouse, C. & Hemler, M. E. (1989) EMBO J. 8, 1361-1368]. Primer extension analysis indicated that alpha 4 mRNA extended well beyond the 5' end of this cDNA. To clone this 5' sequence, a primer extension cDNA library was constructed, and a cDNA extending an additional 660 base pairs was isolated. This cDNA hybridized to multiple mRNAs in both T and B lymphocytes, but no alpha 4 mRNA was found in different tissues or in adherent cell lines. A single alpha 4 gene was detected in a genomic Southern blot when hybridization was done at high stringency; however, additional bands were observed at lower stringency, indicating the presence of alpha 4-related genes. Some of the different mRNAs that hybridize to the alpha 4 cDNA may then be the products of these related genes. Analysis of the alpha 4 genomic sequence revealed a large first exon of 958 base pairs. Interestingly, translation of alpha 4 initiates from the second ATG in this exon (nucleotide + 744). The first ATG (nucleotide +21) is followed by a termination codon 21 amino acids downstream. Such upstream ATG codons have been implicated in translational control of protooncogenes. One major transcriptional start site was identified by using S1 nuclease and primer extension mapping. Consensus sequences for DNA regulatory elements were found upstream of the gene and in exon 1 and intron 1. The alpha 4 gene 5' flanking region acted as a promoter in transfection assays. Detailed characterization of the promoter should provide insight into molecular events regulating expression and tissue specificity of alpha 4.

Abstract

Regulation of cyclooxygenase expression was studied in homogenous preparations of epithelial cells isolated from sheep tracheal mucosa. Cellular capacity to generate cyclooxygenase-derived arachidonate metabolites (predominantly prostaglandin E2) increased markedly in cultured compared to freshly isolated cells. A 70 kDa cyclooxygenase protein and corresponding 2.8 kb mRNA were coordinately expressed but their levels did not increase proportionately to the increase in cyclooxygenase activity. Rehybridization of Northern blots at lower stringency revealed the presence of a new tissue-specific 4.0 kb mRNA species exhibiting increased expression during cell culture. Hybridization of the 4.0 kb mRNA with two nonoverlapping cDNA probes at only low stringency conditions suggests that it is derived from a distinct gene. Its relatedness to cyclooxygenase and its increase in parallel with enzymatic activity further suggest that the larger mRNA may encode for a cyclooxygenase.

Abstract

To determine identities of mediators and mechanisms for their release from pulmonary airway epithelial cells, we examined the capacities of epithelial cells from human, dog and sheep airways to incorporate, release and oxygenate arachidonic acid. Purified cell suspensions were incubated with radiolabeled arachidonic acid and/or ionophore A23187; fatty acid esterification and hydrolysis were traced chromatographically, and oxygenated metabolites were identified using high-pressure liquid chromatography and mass-spectrometry. In each species, cellular uptake of 10 nM arachidonic acid was concentrated in the phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine fractions, and subsequent incubation with 5 microM A23187 caused release of 10-12% of the radiolabeled pool selectively from phosphatidylcholine and phosphatidylinositol. By contrast, the products of arachidonic acid oxygenation were species-dependent and in the case of human cells were also novel: A23187-stimulated human epithelial cells converted arachidonic acid predominantly to 15-hydroxyeicosatetraenoic acid (15-HETE) and two distinct 8,15-diols in addition to prostaglandin (PG) E2 and PGF2 alpha. Cell incubation with exogenous arachidonic acid (2.0-300 microM) led to progressively larger amounts of 15-HETE and the dihydroxy, epoxyhydroxy and keto acids characteristic of arachidonate 15-lipoxygenase. Both dog and sheep cells converted exogenous or endogenous arachidonic acid to low levels of 5-lipoxygenase products, including leukotriene B4 without significant 15-lipoxygenase activity. In the cyclooxygenase series, sheep cells selectively released PGE2, while dog cells generated predominantly PGD2. The findings demonstrate that stereotyped esterification and phospholipase activities are expressed at uniform levels among airway epithelial cells from these species, but pathways for oxygenating arachidonic acid allow mediator diversity depending greatly on species and little on arachidonic acid presentation.